Clarification of transgenic milk using depth filtration

ABSTRACT

Processes and apparati are provided for separating molecules of interest from a mixture by depth filtration (DF). The DF of the invention is useful in the clarification and processing of various feedstreams for the removal of a molecule of interest. According to a preferred embodiment, a transgenic milk feedstream is stabilized and particulate matter such as fat, casein miscelles and bacteria are removed. An aseptic filtration step was also developed to remove any bacteria remaining in a clarified transgenic milk feedstream.

FIELD OF THE INVENTION

The present invention provides an improved method and system ofpurifying specific target molecules from contaminants found in aninitial feedstream. More specifically, the methods of the currentinvention provide for the processing of a sample solution through animproved method of depth filtration that enhances the purification,clarification and fractionation of a desired molecule from a givensource material.

BACKGROUND OF THE INVENTION

The present invention is directed to improved methods and apparati forthe production of proteins of interest from a given source material. Itshould be noted that the production of large quantities of relativelypure, biologically active molecules is important economically for themanufacture of human and animal pharmaceutical formulations, proteins,enzymes, antibodies and other specialty compounds. In the production ofmany polypeptides, antibodies and proteins, various recombinant DNAtechniques have become the method of choice since these methods allowthe large scale production of such proteins. The various “platforms”that can be used for such production include bacteria, yeast, insect ormammalian cell cultures as well as transgenic plants or animals. Fortransgenic animal systems, the preferred animal type is production indairy mammals, but the transgenics platform technology also contemplatesthe use of avians or other animals to produce exogenous proteins,antibodies, or fragments or fusions thereof.

Producing recombinant proteins involves transfecting host cells with DNAencoding the protein of interest and growing the host cells, transgenicanimals or plants under conditions favoring expression of therecombinant protein or other molecule of interest. The prokaryote—E.coli has been a favored cell culture host system because it can be madeto produce recombinant proteins in high yields. However, E. coli areoften unable to produce complex or large molecules with proper tertiaryfolding and resulting in lower or aberrant biological activity.

With improvements in the production of exogenous proteins or othermolecules of interest from biological systems there has been increasingpressure on the biotechnology industry to develop new techniques toenhance the volume of production while simultaneously making it moreefficient and cost effective in terms of the purification and productrecovery. That is, with new products, and larger volumes of knownproducts there is substantial interest in devising methods to bringthese therapeutics, in commercial volumes, to market quickly. At thesame time the industry is facing new challenges in terms of developingnovel processes for the recovery of transgenic proteins and antibodiesfrom various bodily fluids including milk, blood and urine.

Filtration technologies have been major tools in food processing formore than 25 years. The food preparation industry represents asignificant part of the filtration and clarification industryworld-wide. The main applications of filtration processes are in thedairy industry (whey protein concentration, milk proteinstandardization, etc.), followed by beverages (wine, beer, fruit juices,etc.) and egg products. Among the very numerous applications of thecurrent invention on an industrial scale, the clarification of fruit,vegetable and sugar juices by microfiltration also allow the flowdynamics to be both simplified and to enhance the final product quality.

With large scale production it is typically the case that there are morecomplex problems. In addition, there are further challenges imposed interms of meeting product purity and safety, notably in terms of virussafety and residual contaminants, such as DNA and host cell proteinsthat might be required to be met by the various governmental agenciesthat oversee the production of biologically useful pharmaceuticals.

Several methods are currently available to separate molecules ofbiological interest, such as proteins, from mixtures thereof. Oneimportant such technique is affinity chromatography, which separatesmolecules on the basis of specific and selective binding of the desiredmolecules to an affinity matrix or gel, while the undesirable moleculeremains unbound and can then be moved out of the system. Affinity gelstypically consist of a ligand-binding moiety immobilized on a gelsupport. For example, GB 2,178,742 utilizes an affinity chromatographymethod to purify hemoglobin and its chemically modified derivativesbased on the fact that native hemoglobin binds specifically to aspecific family of poly-anionic moieties. For capture these moieties areimmobilized on the gel itself. In this process, unmodified hemoglobin isretained by the affinity gel, while modified hemoglobin, which cannotbind to the gel because its poly-anion binding site is covalentlyoccupied by the modifying agent, is removed from the system. Affinitychromatography columns are highly specific and thus yield very pureproducts; however, affinity chromatography is a relatively expensiveprocess and therefore very difficult to put in place for commercialoperations.

In both the biotech industry and in industry ultrafiltration hastraditionally been used for size-based separation of protein mixtureswherein the ratio of the protein molecular masses have to be at leastaround 10 to 1. This has been a limiting factor in many industrialapplications throughout industry and in particular in the recovery ofbiopharmaceuticals in the milk of transgenic mammals. Significantresearch has taken place in the optimization of ultrafiltration systemsby altering the physiochemical conditions (i.e. pH and ionic strength)to achieve higher selectivities (Van Reis et al. (1997)).

More specifically, depth filtration (DF) and tangential flowmicrofiltration (MF TFF) are two widely adopted filtration techniquesthat are related, but differ in their manipulation of functional flowmechanics. Generally, in DF processes, the feedstream is preferablyintroduced perpendicular to the membrane surface. Substances smallerthan the membrane pores can become trapped either on the membrane'ssurface or within the membrane matrix, whereas the filtrate passesthrough the membrane. Sometimes referred to as “dead-end” or “depth”filtration, DF is commonly used in applications such as clarification,prefiltration, sterile filtration and virus removal. Additionally themajority of depth filters used in the pharmaceutical industry aredisposable in nature.

Alternatively, with MF TFF processes, the feedstream is introducedparallel to the membrane surface, resulting in a continuous sweeping ofthe filtration source material. Under optimal conditions, substancessmaller than the membrane's pores escape as filtrate or permeate, andlarger particles are retained as retentate. Because of MF TFF's sweepingaction and cross-flowing process stream, TFF-based techniques are lessprone to fouling than the DF processes of the invention, in whichseparated particles can accumulate either on or in the membrane. TFFsystems exhibit predictable performance characteristics, reliability,and ability to process “difficult” feed streams—all of which havecontributed to establishing this platform as the preferred separationmethod for many biopharmaceutical applications. TFF systems andmembranes are not disposable, membranes are cleaned between batches andreused. For these reasons MF TFF systems are frequently used to separatesmall molecules (1-1000 kD) from larger particulates (1 um -10 um).However, the energy and cleaning associated with the use of MF TFF canoften make its use in large volume enterprises impractical.

As mentioned, purifying a recombinant protein from milk is technicallycomplex and expensive. The purification process must be reproducible,involving as few labor-intensive steps as possible, and maximize theyield of the target protein as measured by its biological activity. Anideal purification process optimizes yield, keeping manufacturing costslow.

Clearly then, there remains a need for the development of additionallarge scale processes for the optimal purification of proteins out oftransgenic milk or host cell culture systems which address the relevantquantitative and qualitative issues. The present invention addresses andmeets these needs by disclosing a purification process which, in part,relies upon a selective precipitation and depth filtration step whichfacilitates removal of vast quantities of contaminating/impurecompounds, enhancing effectiveness, reducing cost and speeding upprocessing from a given feedstream.

According to the methods of the current invention improvements have beenmade to optimize conditions in order to increase the potential sizeexclusion properties. Various particulates in milk, such as casein andfat, are micelles. These micelles can be manipulated by bufferconditions and be forced to increase or decrease in size. Thismanipulation of buffer is used to increase the separation efficiency ofthe depth filtration process. These processes make possible thedevelopment of high-performance depth filtration (DF) from variousfeedstreams including milk. One molecule of interest that can bepurified from a cell culture broth or a transgenic milk feedstream ishuman recombinant antithrombin. Other molecules of interest includewithout limitation, human albumin, alpha-1-antitrypsin, antibodies, Fcfragments of antibodies and fusion molecules wherein a human albuminprotein acts as the carrier molecule. The resulting DF system isemployed through the current invention to improve clarification andfractionation efforts even from the levels achieved by TFF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Shows the Processing of rhAT according to the Depth Filtrationtechniques of the invention.

FIGS. 2A-2D Show the Volumetric Throughput versus Resistance of thecurrent invention for a feed stream of interest.

FIG. 3 Shows a non-reduced SDS page gel demonstrating the amount of rhATrecovery from a DF Matrix.

FIG. 4 Shows an SDS gel of rhAT recovery using a M403 (5 μm) gradefilter for the depth filtration/clarification of whole milk.

FIG. 5 Shows full Filtration Process Flow Diagram throughmicrofiltration, ultrafiltration and final asceptic filtration. DepthFiltration would occur in the microfiltration step.

FIG. 6 Shows the process of generating a transgenic animal capable ofproducing a protein of interest in their milk.

FIG. 7 Shows a DF System Diagram.

FIG. 8 Shows a Depth Filtration graph of rhAT of Volumetric Throughputversus Resistance with a M103 (10 μm) filter.

FIG. 9 Shows a Depth Filtration graph of rhAT of Volumetric Throughputversus Resistance with a M453 (2.5 μm) filter.

FIG. 10 Shows an SDS gel of rhAT compared to a Dual TFF filtration run.

FIG. 11 Shows an SDS gel of rhAT from a TFF experiment and two differentDF experiments, one control and one heat treated.

FIGS. 12A-12B Shows a SEC Chromatogram of Recovered rhAT.

FIG. 13 Shows an enlarged portion of the SEC Chromatogram of DFrecovered rhAT Compared to Alternative Methods.

FIG. 14 Shows a SEC Chromatogram of DF Recovered rhAT Compared toAlternative MF TFF Methods.

SUMMARY OF THE INVENTION

Briefly stated, the objective of the current invention is to use DepthFiltration (DF) techniques to achieve enhanced clarification andfractionation of a protein of interest. That is, to improve theseparation efficiencies of a protein of interest from an initialfeedstream using DF. More specifically, it is an object of the presentinvention to provide a depth filtration method and a series of reactantsthat substantially increase the effectiveness of such filtering activityfrom milk or cell culture fluid as a starting feedstream.

One protein of interest, and used as an example herein, is recombinanthuman antithrombin. The goal of the methods of the current invention areto pass the target protein, recombinant human antithrombin (rhAT) andretain the major contaminating milk proteins in the most efficientmanner possible. According to the current invention, contaminating milkproteins include IgG, Lactoferrin, albumin, casein, lactoglobulin, andlactalbumin which are removed from a clarified bulk protein of interest.The methods of the current invention use rhAT as an exemplar but can beused for other proteins of interest.

Therefore, in a preferred embodiment of the current invention thefiltration technology developed and provided herein provides a processto clarify and fractionate the desired recombinant protein or othermolecule of interest from the native components of milk or contaminantsthereof. The resulting clarified bulk intermediate is a suitable feedmaterial for traditional purification techniques such as chromatographywhich are used down stream from the DF process to bring the product to afinal formulation and purity useful for medicinal applications.

A preferred protocol of the current invention employs three filtrationunit operations that clarify and fractionate the product from a giventransgenic milk volume containing a molecule of interest. Theclarification step removes larger particulate matter, such as fatglobules and casein micelles from the product. The concentration andfractionation steps thereafter remove most small molecules, includinglactose, minerals and water, to increase the purity and reduce thevolume of the resulting product composition. The product of the DFprocess is tailor concentrated to a level suitable for optimal downstream purification and overall product stability. This clarifiedproduct is then aseptically filtered to assure minimal bioburden andenhance stability of the product for extended periods of time. The bulkproduct will realize a purity between 65% and 85% and may containcomponents such as albumin, whey proteins (β Lactoglobulin, αLactalbumin, and BSA), and low levels of residual fat and casein. Thispartially purified product is an ideal starting feed material forconventional down stream chromatographic techniques.

Typical of the products that the current invention can be used toprocess are other transgenically produced recombinant proteins ofinterest, including without limitation: antithrombin, rhAT, IgG1antibodies, fusion proteins (ex: erythropoietin—human albuminfusion—“HEAP” or Human Albumin—Erythropoietin; or, a β-Interferon—rhAT),alpha-1-antitrypsin, IgG4, IgM, IgA, Fc portions, fusion moleculescontaining a peptide or polypeptide joined to a immunoglobulin fragment.Other proteins that can be processed by the current invention includerecombinant proteins, exogenous hormones, endogenous proteins orbiologically inactive proteins that can be later processed to restorebiological function. Included among these processes, without limitation,are human growth hormone, antichymotrypsin, recombinant human albumin,decorin, human urokinase, tPA and prolactin.

Moreover, according to the current invention the alterations in salt(Ammonium Sulfate or EDTA) concentration differ from the prior art andserve to enhance the purity available according to those using themethods of the current invention.

According to additional embodiments of the current invention the DFtechniques provided herein are applicable to a variety of differentindustries. In the beer industry, recovery of maturation andfermentation tank bottoms is already applied at industrial scale. Duringthe last decade significant progress has been made with microfiltrationmembranes in rough beer clarification. The techniques of the currentinvention may be applicable in these efforts. Relative to wine improvedfiltration technologies will provide for improved microbiological andtartaric stability. In the milk and dairy industry, bacteria removal andmilk globular fat fractionation using enhanced DF microfiltrationtechniques for the production of drinking milk and cheese milk are alsouseful.

It is an object of the present invention to provide more efficient depthfiltration processes for separating species such as particles andmolecules by size, which processes are selective for the species ofinterest, resulting in higher-fold purification thereof.

It is another object to provide improved filtration processes, includingdepth filtration processes, for separating biological macromolecules,such as proteins, from contaminating particles, such as fat and caseinmicelles, which causes pore fouling and flux decay.

These and other objects will become apparent to those skilled in theart. Other features and advantages of this invention will becomeapparent in the following detailed description of preferred embodimentsof this invention, taken with reference to the accompanying drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The following abbreviations have designated meanings in thespecification:

Abbreviation Key:

-   -   BSA Bovine Serum Albumin    -   CHO Chinese Hamster Ovary cells    -   CV Crossflow Velocity    -   DF Depth Filtration    -   DV Diafiltration Volume    -   IEF Isoelectric Focusing    -   GMH Mass Flux (grams/m²/hour)—also J_(M)    -   LMH Liquid Flux (liters/m²/hour)—also J_(L)    -   LPM Liters Per Minute    -   M Molar    -   MF Microfiltration    -   NMWCO Nominal Molecular Weight Cut Off    -   NWP Normalized Water Permeability    -   PES Poly(ether)-sulfone    -   pH A term used to describe the hydrogen-ion activity of a        chemical or compound according to well-known scientific        parameters.    -   PPM Parts Per Million    -   SDS-PAGE SDS (sodium dodecyl sulfate) Poly-Acrylamide Gel        electrophoresis    -   SEC Size Exclusion Chromatography    -   TFF Tangential Flow Filtration    -   PEG Polyethylene glycol    -   TMP Transmembrane Pressure    -   UF Ultrafiltration

Explanation of Terms: Clarification

The removal of particulate matter from a solution so that the solutionis able to pass through a 0.2 μm membrane.

Colloids

Refers to large molecules that do not pass readily across capillarywalls. These compounds exert an oncotic (i.e., they attract fluid) loadand are usually administered to restore intravascular volume and improvetissue perfusion.

Concentration

The removal of water and small molecules with a membrane such that theratio of retained molecules to small molecules increases.

Concentration Polarization

The accumulation of the retained molecules (gel layer) on the surface ofthe membrane caused by a combination of factors: transmembrane pressure,crossflow velocity, sample viscosity, and solute concentration.

Darcy's Law

An empirical law that governs flow through a porous media and alsodescribes the relationship among flow rate, pressure drop, andresistance. Filter aid products are usually processed to provide a rangeof filtration rates that are closely related to permeability as reportedin Darcy units.

Depth Filtration

A treatment process in which the entire filter bed is used to trapinsoluble and suspended particles in its voids as water flows throughit. The three dimensional sample collection patch may include a materialcapable of providing depth filtration or sieve filtration of a sample.Capacity is determined by the depth of the matrix. In depth filtration,particulates are trapped both within the matrix and on the surface ofthe filtration medium.

Diafiltration

A fractionation process of washing smaller molecules through a membrane,leaving the larger molecule of interest in the retentate. It is aconvenient and efficient technique for removing or exchanging salts,removing detergents, separating free from bound molecules, removing lowmolecular weight materials, or rapidly changing the ionic or pHenvironment. The process typically employs a microfiltration membranethat is employed to remove a product of interest from a slurry whilemaintaining the slurry concentration as a constant.

Feedstream

The raw material or raw solution provided for a process or method andcontaining a protein of interest and which may also contain variouscontaminants including microorganisms, viruses and cell fragments. Apreferred feedstream of the current invention is transgenic milkcontaining a exogenous protein of interest.

Filter Cake

Retained solids and filter media on the filter element.

Filtrate Flux (J)

Represents the rate at which a portion of the sample has passed throughthe membrane.

Flow Velocity (V)

The speed at which the fluid passes the surface of the membrane isconsidered the fluid flow velocity. Product flux will be measured asflow velocity is varied. The relationship between the two variables willallow us to determine an optimal operational window for the flow.

Fractionation

The preferential separation of molecules based on a physical or chemicalmoiety.

Gel Layer

The microscopically thin layer of molecules that can form on the top ofa membrane. It can affect retention of molecules by clogging themembrane surface and thereby reduce the filtrate flow.

Membrane Pore Size Rating (MPSR)

A membrane pore size rating, typically given as a micron value,indicates that particles larger than the rating will be retained by themembrane.

Nominal Molecular Weight Cut Off (NMWCO)

The size (kilodaltons) designation for the ultrafiltration membranes.The NMWCO is defined as the molecular weight of the globular proteinthat is 90% retained by the membrane.

Nominal Molecular Weight Limits (NMWL)

A membrane rating system that indicates that most dissolvedmacromolecules with molecular weights higher than the NMWL and some withmolecular weights lower than the NMWL will be retained by the membranein question.

Normalized Water Permeability (NWP)

The water filtrate flow rate established at a specific recirculationrate during TFF device initial cleaning. This value is used to calculatemembrane recovery.

Microfiltration

Microfiltration is a pressure-driven solid-liquid separation process.According to the invention, microfiltration techniques are capable ofremoving suspended solids in the 0.10-1.0 micron range. In comparison,ultrafiltration is generally used with solids in the 0.01-0.10 micronrange.

Molecule of Interest

Particles or other species of molecule that are to be separated from asolution or suspension in a fluid, e.g., a liquid. The particles ormolecules of interest are separated from the fluid and, in mostinstances, from other particles or molecules in the fluid. The size ofthe molecule of interest to be separated will determine the pore size ofthe membrane to be utilized. Preferably, the molecules of interest areof biological or biochemical origin or produced by transgenic or invitro processes and include proteins, peptides, polypeptides, antibodiesor antibody fragments. Examples of preferred feedstream origins includemammalian milk, mammalian cell culture and microorganism cell culturesuch as bacteria, fungi, and yeast. It should also be noted that speciesto be filtered out include non-desirable polypeptides, proteins,cellular components, DNA, colloids, mycoplasm, endotoxins, viruses,carbohydrates, and other molecules of biological interest, whetherglycosylated or not.

Precoat

A precoat is a thin layer, typically between 1.5 to 3.0 mm, of a filteraid that is applied to the septum before the actual filtration process.A precoat is usually unnecessary when using a depth filter as the septum

Tangential Flow Filtration

A process in which the fluid mixture containing the components to beseparated by filtration is re-circulated at high velocities tangentialto the plane of the membrane to increase the mass-transfer coefficientfor back diffusion. In such filtrations a pressure differential isapplied along the length of the membrane to cause the fluid andfilterable solutes to flow through the filter. This filtration issuitably conducted as a batch process as well as a continuous-flowprocess. For example, the solution may be passed repeatedly over themembrane while that fluid which passes through the filter is continuallydrawn off into a separate unit or the solution is passed once over themembrane and the fluid passing through the filter is continuallyprocessed downstream.

Recovery

The amount of a molecule of interest that can be retrieved afterprocessing. Usually expressed as a percentage of starting material oryield.

Retentate

The portion of the sample that does not pass through the membrane, alsoknown as the concentrate. Retentate is being re-circulated during theTFF.

The biologics industry is becoming increasingly concerned with productsafety and purity, as well as cost of goods. The use of DF, according tothe current invention, is a rapid, cheaper and more efficient method forbiomolecule separation. It can be applied to a wide range of biologicalfields such as immunology, protein chemistry, molecular biology,biochemistry, and microbiology.

It should also be noted that genetically engineered biopharmaceuticalsare purified from a supernatant containing a variety of diverse hostcell contaminants. Reversed-phase high-performance liquid chromatography(RP-HPLC) is another method that can be used for protein purificationbecause it can efficiently separate molecular species that areexceptionally similar to one another in terms of structure or weight.Procedures utilizing RP-HPLC have been published for many molecules.McDonald and Bidlingmeyer; “Strategies for Successful Preparative LiquidChromatography” PREPARATIVE LIQUID CHROMATOGRAPHY, Brian A. Bidlingmeyer(New York: Elsevier Science Publishing, 1987), vol. 38, pp. 1-104; Leeet al., Preparative HPLC. At the 8TH BIOTECHNOLOGY SYMPOSIUM, Pt. 1,593-610 (1988). However, at commercial scale RP-HPLC is neither ascost-efficient nor as effective of the current invention.

The current invention provides the results of clarifying transgenic goatmilk using “dead end” filtration or “depth filtration.” Until the use ofthese specific buffer salts in milk, it could not be effectivelyclarified using DF because the size of the casein micelle was too smallto be retained by these coarse filters. Fine filters able to retain thecasein were plugged rapidly and not able to be used effectively.According to a preferred embodiment of this invention the milk of atransgenic dairy animal, a goat, was purified to a clarified bulkmaterial using depth filtration. Depth filtration's advantage overtangential flow filtration is that no recirculation is needed forprocess filtration. The liquid is simply pumped in through the systemand the filtrate exits the designated filter downstream. The filterelements are then disposed of eliminating the need for cleaning of thefilters. Additionally milk clarified using depth filtration is producedin a single pass as opposed to lengthy recirculation process required bytangential flow filtration or other filtration schemes. Similar uses ofthe embodiments of the current invention could also be applied toisolating a protein of interest from a cell culture feedstream.

Process Steps

Processing steps from transgenic mammals include the following:

-   -   1. Collection Milk collection from each animal    -   2. Dilute the milk 1:1 with 3.8M Ammonium Sulfate    -   3. Perform depth filtration/0.2 um Aseptic filtration

Collect Permeate

The present invention particularly contemplates filter applications of atype wherein the filter media is generally not reusable but is discardedtogether with the particulate solids removed from the fluid beingfiltered. Since the particulate solids represent a necessary disposalcomponent, the total amount of solids to be disposed of from thefiltering application can best be minimized by reducing the amount offilter media accompanying the particulate solids, and/or increasing theamount of solids retained per unit volume of filter media.

For filter applications of the type referred to above, filter media haslong been employed wherein relatively thin and open wet strength layersare arranged on opposite surfaces of the filter media. The relativelythin and open structure of the wet strength layers are desirable forpermitting maximum flow of fluid to be filtered through the filtermedia. Typically, one or more layers of filter septum material have beenarranged between the wet strength layers to achieve depth filtration asdescribed above. Furthermore, the wet strength layers have typicallybeen bonded to the filter septum material, preferably by binder oradhesive which is commonly sprayed onto a surface of the filter septummaterial. The wet strength layer is then pressed onto the filter septummaterial in order to bond the two layers together. Bonding of the layersis generally necessary to maintain continuity of the filter media, forexample, when it is replaced in the filter apparatus. The wet strengthlayer, by itself, is typically quite open and presents very littleinterference to the flow of liquid to be filtered through the filtermedia. However, the manner in which binder is commonly applied to bondthe wet strength layer to the filter septum material typically resultsin the binder itself being a much greater cause of blinding or flowreduction than the wet strength layer itself.

Basics of Depth Filtration

Generally, a depth filter media is one having substantial tortuous pathswhich are capable of receiving and retaining smaller particulatematerial upon and within the cross-section of the filter media itself.Preferably, the depth filter media is formed with a matrix ofmulti-directional fibers forming the tortuous passages so that they arecapable of trapping and retaining the smaller particles. A depth filtermedia accomplishes filtration at least partly because fluid passingthrough the filter media is caused to change direction as it passesthrough the multi-directional fibers. This in turn causes very fineparticulate material in the liquid to be deposited and retained inniches or crevices even though the particles may be smaller than theopenings in the media.

A depth filtration process is provided herein to remove cell debris,insoluble contaminating milk proteins, fat, and nucleic acidprecipitate. This step provides a convenient means to economicallyremove cell debris, contaminating proteins and precipitate. In choosinga filter or filter scheme it was necessary to ensure a robustperformance in the event upstream changes or variations occur.Maintaining the balance between good clarification performance and stepyield requires investigation of a large variety of filter types withvarying internal media. Suitable filters may utilize cellulose filters,regenerated cellulose fibers, cellulose fibers combined with inorganicfilter aids (e.g. diatomaceous earth, perlite, fumed silica), cellulosefibers combined with inorganic filter aids and organic resins, or anycombination thereof, and polymeric filters (examples include but are notlimited to nylon, polypropylene, polyethersulfone) to achieve effectiveremoval.

Depth Filtration

Depth Filtration is a treatment process in which the entire filter bedis used to trap insoluble and suspended particles in its voids as waterflows through it. The sample collection patch may include a materialcapable of providing depth filtration of a sample. In depth filtration,particulates are trapped both within the matrix and on the surface ofthe filtration medium. Depth filters are composed of random mats ofmetallic, polymeric, inorganic, or organic materials. Depth filters relyon the density and thickness of the mats to trap particulates andfluids, and generally retain large quantities of particulates or fluidswithin the matrices. Certain disadvantages of depth filters includemedia migration, which is the shifting of the filter medium understress, and particulate unloading at high differential pressures.Advantages of depth filters include reduced cost, high throughputs, highvolume-holding capacity, removal of a range of particle sizes, and highflow rates. The extract of intracellular molecules is then separatedfrom the remaining insoluble slurry by depth filtration, for example,using diatomaceous earth in a plate and frame filter press.

With a depth filter media as contemplated by the present invention, thefilter media has or forms passages throughout its matrix which arecapable of trapping and retaining very small particles, preferably inthe range of about 1-5 microns.

The compositions for filtrations vary. In rough filtration or largevolume filtration applications loose media such as diatomaceous earth(body remnants of extinct animals called diatoms) and/or perlite (aground volcanic glass), have been used as depth filter media in pressureleaf filters. In the past cellulose, asbestos or other synthetic fibershave been combined with such loose media or used as pre-coatingmaterials to prevent migration of the filter aid particulates throughthe filter screen support. Porous cellulose fiber membranes, ceramicmembranes, wet strength resin binders and dry strength resin bindershave also been used.

In order to assure continued effectiveness of the depth filtrationtechniques of the invention, it is also important that the filter mediaremain open at its top surface or, in other words, that it not beblinded by components of the filter media itself such as the wetstrength layer or an associated binder or by particulate materialdeposited from the liquid being filtered. The present invention noveltyassures that the depth filter media remains open by avoiding the use ofadhesive or wet strength material formed on the top surface of thefilter media receiving the liquid to be filtered. Accordingly, it isparticularly important to understand that depth filtration isaccomplished by the filter septum layer of the present invention andthat the top surface of the filter septum layer itself remains exposedfor receiving the liquid to be filtered.

As for particle size, depth filtration is generally contemplated forpurposes of the present invention to include applications where theminimum particulate size is about 50 microns or less, usually with asubstantial portion of the particulate solids being smaller than 50microns. More preferably, depth filtration is contemplated for thepresent invention where particulate solids have a minimum size in therange of about 1-25 microns. As will be apparent from the followingdescription, the depth filter media of the present invention isparticularly useful for removing a substantial portion of thoseparticulate solids.

According to the invention, there are at least two important conceptscooperating to form the foundation of the current invention. The firstinvolves disassociating the casein micelles in milk with EDTA andclarifying the feed stream with a depth filter. The second involvesaggregating the casein micelles with ammonium sulfate and clarifying themilk using a depth filter. The technology is a new process that combinesexisting methods in a novel manner producing a favorable result. The useof depth filtration to clarify milk did not seem feasible until the milkwas treated with a buffer to alter the state of the casein micelle.Preferred embodiments of the current invention can be made operationalin a closed system with simple skid design greatly enhancing the abilityof users to maintain Good Manufacturing Processes (“GMP”) operationsand/or the containment of bio-hazardous agents. The process is alsoquicker and less labor intensive. Typically, flux rates for thefiltration are between 60-80LMH and between 30-40 liters/m2 of milk canbe processed in approximately 2 hours.

The resulting permeate consists of the clarified milk which containsvarious soluble milk proteins and the transgenic protein of interest.The resulting retentate (or cake layer) which consists of a suspensionof insoluble proteins and fat may be washed or solubilized and passedthrough the depth filter for collection. The remaining cake layercontains the remaining insoluble components of milk which is usuallydiscarded.

Filtration methods of the type contemplated by the present invention areperformed in filtration apparatus. The filter apparatus is commonlyreferred to as a filter press and includes relatively movable filterplates. One of the filter plates is connected with an inlet forreceiving fluid to be filtered. Typically, the fluid is a liquid andeven more typically water or water based fluids containing particulatesolids to be removed during the filtration process. The other filterplate is connected with an outlet for receiving fluid passing throughthe filter apparatus and having particulate solids removed. Thus, thefiltered fluid may be disposed of or recycled for further use, dependingupon the particular application in which the filter apparatus isemployed.

For replacement of the filter media, the flow of fluid through thefilter apparatus is temporarily interrupted, the filter assembly isvoided of fluid and the filter plates are separated from each other. Thefilter media is then withdrawn from the filter apparatus. At the sametime, a fresh surface portion of the filter media is drawn, for example,from the supply roll into the filter apparatus. At that time, the filterplates are again pressed into engagement with each other to capture andseal the fresh supply of filter media there between and the filtrationoperation continued with the flow of additional fluid from the source.

Suitable microfiber materials according to the present invention includeglass, polyester, polypropylene, polyethylene, nylon and other syntheticfibers having generally similar characteristics. It is generallybelieved that all of these synthetic fibers are available in both theshort and long lengths described above. Typically, the synthetic fibersare relatively straight and round while normally resisting absorption ofliquids because of their synthetic composition.

Once the precipitation is complete, depth filtration is conductedsequentially on each aliquot using the same filtration apparatus. Itwill become evident upon review of this specification that the processesof the present invention are scaleable, running the gamut from smallerscale (e.g., about 5-10 liter runs) all the way to commercial scalepreparations, such as 1,000 to 5,000 L production runs. According to thecurrent invention the process will be linearly scalable. The initialprocess steps (precipitation, depth filtration, and ultrafiltration)scale with feedstream volume while the anion exchange chromatography andsubsequent steps scale with viral particle input.

Sterile filtration may be added to the current process to eliminatebioburden. The sterile filter may be constructed of a variety of othermaterials that are well known in the art and available to the artisan.These may include, but are not limited to, polypropylene, cellulose,regenerated cellulose, cellulose esters, nylon, polyethersulfone, or anyother material which is consistent with low product binding. The filtermay have a single membrane layer or may incorporate a prefilter of thesame of different material. The product can be held frozen or atapproximately 4° C. for subsequent formulation and filling.

According to another embodiment an orthogonal purification step may alsobe added to deal with impurity clearance, as well as an adventitiousagent clearance step. Orthogonal purification steps are not necessarilyrequired and may be assessed by the skilled artisan and in turnimplemented based on need. Potential steps include flow-through cationexchange chromatography, reversed-phase adsorption, and hydroxyapatitechromatography. An anion exchange chromatography step also can beconsidered for removing additional impurities. This step can be operatedin either bind/elute or flow-through modes. The step can be placed aftereither ultrafiltration step by ending the UF with a diafiltration intoan appropriate buffer such as phosphate buffered saline (PBS).

According to the current invention a recombinant protein, rhAT, isselected for use in the development of new clarification techniques.This protein is expressed in transgenic milk that must be clarifiedprior to purification. Depth Filtration using depth filter media offersan attractive alternative to centrifugation or tangential flowfiltration. It is simple to use, has low initial costs associated withset-up, and is disposable. The objective of milk clarification/asepticfiltration is to isolate the soluble components of milk, called wheyproteins, and render a microbiologically stable product. The wheyproteins include IgG, Lactoferin, Albumin, residual soluble Casein,Lactoglobulin, Lactalbumin, and the rhAT recombinant protein. The milkalso contains particulate matter like fat globules, casein micelles andcell debris. Looking to FIG. 1, the particulates can be separated fromthe whey proteins, once the precipitation is complete, by passing themthrough a 5 μm depth filter.

According to a preferred embodiment of the current invention a criticalstep in clarifying a milk feedstream using depth filters is to partiallyprecipitate the casein micelles using Ammonium Sulfate. In the presenceof 1.9M Ammonium Sulfate at pH 6.5, the casein begins to aggregate andthe aggregate is easily retained by the more open depth filters.Additionally other less soluble milk proteins precipitate, IgG as anexample is also removed using this method. Lastly, fat globules and celldebris are easily removed by the filter, yielding clarified wheyproteins in the filtrate. According to the invention, the filtrate maythen be aseptically filtered and stored at 4° C. prior to purification.

According to one embodiment of the current invention the DF techniquesof the invention are provided followed by enhanced purificationtechniques leading to a pharmaceutical grade therapeutic compositionthat is bioactive. This process can be accomplished by furtherclarifying a DF fractionated feedstream by using a series of ionexchange chromatography columns. Such columns will preferably contain anexchange resin but may also provide for an affinity resin withcontaminants being either captured on additional columns or washed away.The molecule of interest is then collected and prepared for delivery.

According to preferred embodiments of the current invention, DFprocessing runs were conducted using a 90 mm test cell and disk offiltration media. The initial experiments scouted four different gradesof media ranging from 2.5 um up to 15 um in “pore size”. Once an idealgrade of media was selected, the clarified milk was purified and used inthe first stage of the purification process at a reduced scale. Theresults from this portion of the experiment confirm the technology iscomparable to the more conventional clarified milk using tangential flowfiltration.

Materials and Methods Materials Description Part Number 90 mm Test Cellw/ pressure gauge M90 PD Masterflex L/S Pump (10-600RPM) 07524-40Masterflex #14 Silicone Tubing 96420-14 Ertel Elsop Filter Media - 2.5um M453 Ertel Elsop Filter Media - 5 um M403 Ertel Elsop Filter Media -10 um M103 Ertel Elsop Filter Media - 15 um M053  2 mm Scrim Pre-FilterN/A 3.8M Ammonium Sulfate, 0.2M Sodium pH 6.5 Phosphate,

Transgenic Goat Milk

Transgenic milk was separately collected from each of the transgenicgoats and held at 4-8° C. until clarifying (<4 days unless noted).

Goat Number Collection Date Storage Temp. G0881 August - December 20054-8° C. G0737 August - December 2005 4-8° C. C248 August - December 20054-8° C. C239 August - December 2005 4-8° C. B121 August - December 20054-8° C.

Clarification—Media Selection

Four separate clarification experiments were performed using DepthFiltration (DF) under similar operating parameters. The purpose of thisexperiment was to see the effect of different media grades onclarification. Initially 150 ml of milk was added to the sanitized feedreservoir and 150 ml of 3.8M Ammonium Sulfate was added to the sanitizedbuffer reservoir of the microfiltration (MF) system. A dual head pumpand static mixer was then used to mix the two streams in equal ratios.The mixture was then passed through the selected grade of media and thefiltrate collected. The final clarified milk was aseptically filteredand stored in a PETG bottle at 4° C.

Analytical Methods

Reverse Phase HPLC(RPC)

-   -   Reverse phase chromatography was performed on each of the        samples to evaluate the rhAT protein concentration isolated by        this process step.

SEC HPLC(RPC)

-   -   Size exclusion chromatography was performed on each of the        samples to evaluate the amount of rhAT monomer in each sample.

Non Reduced SDS PAGE (RPC)

-   -   SDS PAGE was performed on each of the samples to evaluate the        clarified milk protein composition.

Results Clarification—Media Selection

Clarification of rhAT transgenic milk was performed using differentdepth filter media between 2.5 um and 15 um under similar flowconditions. The pressure profiles for each run may be seen below ingraphs 1-4. Starting in the upper left working down the page the resultsof throughput vs. resistance may be seen. As expected the resistancerises more quickly in the tightest grade of filter, M453 (2.5 um). Thelowest resistance may be seen when using the M053 (15 um). Additionallythe highest filtrate clarity was observed in the M453 (2.5 um) and thelowest in the M053 (15 um). All clarified milk samples were asepticallyfiltered using an 0.2 um syringe filter and the throughput recorded. Asexpected the clearest MF permeate had the highest throughput. It was forthis reason that the M403 (5 um) grade filter was chosen as it had thebest balance between clarity and throughput.

Milk Composition:

Cow milk is about 87% water, 4-5% fat, 5% carbohydrate, and 3-4%protein. Goat's milk and sheep's milk have lower fat content but higherprotein content. Lactose is the major carbohydrate in the milk of mostspecies—and the least variable component of milk. The fat component is acomplex mixture of lipids secreted as globules primarily composed of atriglyceride surrounded by a lipid bilayer membrane, which helps tostabilize those fat globules in an emulsion within the aqueousenvironment of milk. More than 95% of total milk lipids are in the formof globules ranging from 0.1 to 15 μm in diameter. These liquid fatdroplets are covered by a thin membrane, 8-10 nm thick, with propertiescompletely different from both milk fat and plasma. The native fatglobule membrane (FGM) is an apical plasma membrane of the secretorycell that continually envelopes the lipid droplets as they pass into thelumen. The major components of that native FGM, therefore, are proteinand phospholipids. The major milk protein is casein. The principalcasein fractions are (s1) and (s2) caseins, -casein, and -casein. Thedistinguishing property of all caseins is their low solubility at pH4.6. A common compositional factor is that caseins are conjugatedproteins, most with phosphate group(s) esterified to serine residues.Most if not all are found within a structure called a micelle. Itsbiological function is to carry large amounts of highly insolublecalcium phosphate to mammalian young in liquid form and to form a clotin the stomach for more efficient nutrition. Micelles are colloidalmolecules with hydrophobic cores and casein-enriched surfaces heldloosely together by calcium phosphate molecules. They form largeaggregates with diameters of 90-150 nm. These aggregates are porousstructures occupying about 4 mL/g and 6-12% of the total volume fractionof milk. The micelle structure also contains minerals, amino acids, andbioactive peptides.

Whey proteins also include a long list of enzymes, hormones, growthfactors, nutrient transporters, and disease-resistance factors. If theproduct protein tends to associate with either the fat or micelles,purification may be simplified, but this scheme is rare. Caseinmolecules can be separated from whey by precipitating out the caseinwith acid (a slow addition of 0.1-N HCl to lower the milk pH to 4.6) orby disrupting the micellar structure using partial hydrolysis of theprotein molecules with a proteolytic enzyme such as chymosin. However,those methods can result in product losses as high as 40-60%, leading tosignificantly lower overall yields (5-25%) and low biological activity.In contrast salt solutions used in this method precipitate the caseinmicelles while not creating product loss provided that the protein ofinterest remains soluble.

Milk as a Feedstream

Milk may be the product of a transgenic mammal containing abiopharmaceutical or other molecule of interest. In a preferredembodiment the system is designed such that it is highly selective forthe molecule of interest. The clarification step removes largerparticulate matter, such as fat globules and casein micelles from themilk feedstream. The concentration/fractionation steps remove most smallmolecules, including lactose, minerals and water, to increased purityand reduce volume of the product. The product of the DF process isthereafter concentrated to a level suitable for optimal downstreampurification and overall product stability. This concentrated product,containing the molecules of interest, is then aseptically filtered toassure minimal bio-burden (i.e., endotoxin) and enhance the stability ofthe molecules of interest for extended periods of time. According to apreferred embodiment of the current invention, the bulk product willrealize a purity between 35% and 65% and may contain components such asgoat antibodies (from transgenic goats), whey proteins (β Lactoglobulin,α Lactalbumin, and BSA), as well as low levels of residual fat andcasein. This partially purified product is an ideal starting feedmaterial for conventional downstream chromatographic techniques tofurther select and isolate the molecules of interest which couldinclude, without limitation, a recombinant protein produced in the milk,an immunoglobulin produced in the milk, or a fusion protein.

According to the current invention the objective of separating theprotein of interest from contaminating proteins using DF isdemonstrated. The goal of this clarification is to retain the fat,casein, and unwanted precipitated proteins while passing the solubleproduct and milk proteins. The RPC and SDS gel results conclusively showthe contaminating milk components can be effectively reduced during theclarification. All but the product and soluble milk proteins areeffectively removed using the 5 μm DF filter and methods of theinvention.

Membrane Pore Size Rating (MPSR)

The DF pore size and milk buffer condition play a considerable role inthe effectiveness of the clarification. Several DF pore sizes wereevaluated including a 2.5 μm, 5.0 μm, 10 μm, and 15 μm. If the pore sizeis to low as in the case of the 2.5 μm, the throughput and flux arereduced, however, the clarity of the permeate was good. Each larger poresize was evaluated for its permeate quality, throughput and flux. The2.5 μm, 5.0 μm, 10 μm filters all proved to retain the majority of thefat and precipitated milk components. The 15 μm filter could notefficiently be used for this filtration as the quality of the permeatewas lower and a portion of the fat passed through the membrane creatinga hazy permeate. The 5.0 μm filter showed the best throughput, flux, andpermeate quality both initially and after being optimized. The pore sizeof this filter proved to be the largest size able to be used, yet stillbe able to retain the insoluble milk contaminants.

Turning to FIGS. 2A-2D, they demonstrate the Resistance of the filterand the volumetric throughput according to preferred embodiment of theinvention.

Turning to FIG. 3, it provides a non-reduced SDS page confirms themajority of rhAT is recovered in the filtrate of each of the experimentspursuant to the current invention. Also worth noting is the similaritybetween each of the filtrate streams (FIG. 3).

Clarification/Purification

Turning to FIG. 4, once the M403 (5.0 μm) grade filter was chosen forthe depth filtration/clarification of whole milk, several batches werepooled, concentrated, and diafiltered using a 30 kD ultrafiltrationmembrane. This pooled sample was then purified using a 16 ml heparincolumn. Clarified milk from the present 500K clarification and 0.1 umdual TFF process were compared as well. In FIG. 11, the SDS PAGE showsthat lanes 2, 7, and 11 contain the purified rhAT elution fraction. Ascan be seen each is similar in composition with the exception of lane 11where aggregate can be seen. This aggregate was most evident in the 0.1um dual TFF sample.

It has been shown in the data for the invention provided herein thatclarification using depth filtration is feasible and can optimize thepurification of several molecules from a milk feedstream. It is notedthat according to preferred embodiments of the current invention theprocesses are scalable, inexpensive, and have a sustained high productyield (>90%).

Other data indicate that scale up from the 90 mm disk to a 1.1 ft²lenticular cartridge enhances the efficiency of the overall process.This cartridge will have the potential to clarify 3 liters of wholemilk.

Milk Processing

According to the methods of the current invention it is preferable ifthe temperature of the milk is raised to 15-25° C. after it is pooled.The milk is pooled in a reservoir and an equal volume of salt buffer isprepared in a second reservoir. Next the two reservoirs are connected toa pump and in line static mixer. Once the feed stream line is connectedto the filter element, the pump is turned on blending the milk andbuffer prior to filtration. The use of the static mixer is significantas the mixing and resonance time is uniform throughout the entirefiltration. Mixing the milk and buffer in a bulk tank prior tofiltration is less desirable as the time that the mixture rests prior tofiltration is variable. The pump is adjusted to the desired flow ratebased on the average flux of 50LMH. After 5 minutes the initial permeatesample(s) are taken and the critical pressures and flow rates areverified. The DF is run at 50LMH with less than 15 psi of transmembranepressure throughout the filtration. The temperature of the milk shouldremain at 20° C.±5. Once the entire volume of milk and buffer areconsumed a volume of buffer, equal to 10% of the starting milk volume,is flushed through the filter to ensure the majority of the solubleprotein is passed to the permeate.

Once filtration is complete, the permeate collection vessel isdisconnected, the filters are disposed of, and the system is drained andcleaned. The DF clarified permeate is aseptically filtered, and storedat 4° C. prior to downstream purification.

Transgenic Animal Production

Other issues affect the overall yield of any manufacturing processinvolving transgenic mammals: stability of constructs, control ofexpression, and seasonal variations in lactation, to name a few. A soundunderstanding of the health and physiology of the livestock species usedis essential because a transgenic animal may live for 7-10 years andwill experience physiological changes and various environmentsthroughout its life as it develops, gives birth, and ultimatelylactates. Recovery processes must be robust enough to handle thosechanges, but if the advances in our understanding of milk compositionand bioprocessing techniques continue, such challenges should beovercome as well.

According to the current invention, to extract a molecule of interestout of a given feedstream in preparation for use by an end use could beconcluded with a series of additional purification steps. In general, amultiple stage process is preferable but not required. An exemplary twoor three-stage process would consist of a coarse filter(s) to removelarge precipitate and cell debris followed by polishing second stagefilter(s) to with nominal pore sizes greater than 0.2 micron but lessthan 1 micron. The optimal combination will be a function of theprecipitate size distribution as well as other variables. In addition,single stage operations employing a relatively tight filter orcentrifugation may also produce a product of good quality. Moregenerally, any clarification approach including dead-end filtration,microfiltration, centrifugation, or body feed of filter aids (e.g.diatomaceous earth) in combination with dead-end or depth filtration,which provides a filtrate of suitable clarity to not foul the membraneand/or resin in the subsequent steps, will be acceptable to practicewithin the present invention.

Cleaning and Storing Protocols

Cleaning the DF filters is not required as they are disposable; one ofthe obvious advantages over re-usable systems that require a largevolume of cleaning solution and water. Once the filtration is completethe filter elements are removed and discarded. The filter housing, feedvessel, and pump are then the only components requiring cleaning. Athirty (30) minute cycle of 0.5M sodium hydroxide followed by 0.3Mcitric acid have proven to be effective when cleaning stainless steelcomponents in the dairy industry. Once the cleaning cycle is complete,the components are rinsed with water and the filters are re-installedprior the next use.

Recombinant Production

A growing number of recombinant proteins are being developed fortherapeutic and diagnostic applications. However, many of these proteinsmay be difficult or expensive to produce in a functional form and/or inthe required quantities using conventional methods. Conventional methodsinvolve inserting the gene responsible for the production of aparticular protein into host cells such as bacteria, yeast, or mammaliancells, e.g., COS or CHO cells, and then growing the cells in culturemedia. The cultured cells then synthesize the desired protein.Traditional bacteria or yeast systems may be unable to produce manycomplex proteins in a functional form. While mammalian cells canreproduce complex proteins, they are generally difficult and expensiveto grow, and often produce only mg/L quantities of protein. In addition,non-secreted proteins are relatively difficult to purify fromprocaryotic or mammalian cells as they are not secreted into the culturemedium.

In general, the transgenic technology features, a method of making andsecreting a protein which is not normally secreted (a non-secretedprotein). The method includes expressing the protein from a nucleic acidconstruct which includes:

-   -   (a) a promoter, e.g., a mammary epithelial specific promoter,        e.g., a milk protein promoter;    -   (b) a signal sequence which can direct the secretion of a        protein, e.g. a signal sequence from a milk specific protein;    -   (c) optionally, a sequence which encodes a sufficient portion of        the amino terminal coding region of a secreted protein, e.g., a        protein secreted into milk, to allow secretion, e.g., in the        milk of a transgenic mammal, of the non-secreted protein; and    -   (d) a sequence which encodes a non-secreted protein,        -   wherein elements (a), (b), optionally (c), and (d) are            preferably operatively linked in the order recited.

In preferred embodiments: elements a, b, c (if present), and d are fromthe same gene; the elements a, b, c (if present), and d are from two ormore genes. In preferred embodiments the secretion is into the milk of atransgenic mammal.

In preferred embodiments: the signal sequence is the β-casein signalsequence; the promoter is the β-casein promoter sequence.

In preferred embodiments the non-secreted protein-coding sequence: is ofhuman origin; codes for a truncated, nuclear, or a cytoplasmicpolypeptide; codes for human serum albumin or other desired protein ofinterest.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare described in the literature.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of understanding, it willbe apparent to those skilled in the art that certain changes andmodifications may be practiced. Therefore, the description and examplesshould not be construed as limiting the scope of the invention, which isdelineated by the appended claims.

Accordingly, it is to be understood that the embodiments of theinvention herein providing for an improved method of depth filtration togenerate a high yield of a molecule of interest from a given feedstreamare merely illustrative of the application of the principles of theinvention. It will be evident from the foregoing description thatchanges in the form, methods of use, and applications of the elements ofthe disclosed may be resorted to without departing from the spirit ofthe invention, or the scope of the appended claims.

PRIOR ART CITATIONS INCORPORATED BY REFERENCE

-   1. Aravindan G R, et al., (1997), Identification, Isolation, and    Characterization Of A 41-Kilodalton Protein From Rat Germ    Cell-Conditioned Medium Exhibiting Concentration-Dependent Dual    Biological Activities, ENDOCRINOLOGY 138(8):3259-68.-   2. Bracewell D. G. et al., (2004) Addressing a Whole Bioprocess in    Real-Time Using an Optical Biosensor-Formation, Recovery and    Purification of Antibody Fragments From a Recombinant E. Coli Host,    BIOPROCESS BIOSYST ENG. 2004 July; 26(4):271-82. Epub 2004 May 5.-   3. Charlton H. R., et al., (1999) Characterization of a Generic    Monoclonal Antibody Harvesting System for Adsorption of DNA by Depth    Filters and Various Membranes, BIOSEPARATION. 8(6):281-91.-   4. Christy C., et al., (2002) High Performance Tangential Flow    Filtration: A Highly Selective Membrane Separation Process,    DESALINATION, Vol. 144: 133-36.-   5. De Jonge, E. et al. (1993), Filtration Processes in the Cohn    Fractionation Process. BIOTECHNOL BLOOD PROTEINS, 227:49-54.-   6. Gabler et al., (1987), Principles of Tangential Flow Filtration:    Applications to Biological Processing, in FILTRATION IN THE    PHARMACEUTICAL INDUSTRY, pp. 453-490.-   7. Ghosh R, et al., (2003) Parameter Scanning Ultrafiltration: Rapid    Optimisation of Protein Separation, BIOTECHNOL BIOENG., March 20;    81(6):673-82.-   8. Koros, W. J. et al., (1996), Terminology for Membranes and    Membrane Processes (IUPAC Recommendations 1996). PURE & APPL. CHEM.    68: 1479-89.-   9. Millesime L, et al., (1996) Fractionation of Proteins with    Modified Membranes, BIOSEPARATION, June; 6(3):135-45.-   10. Morcol et al., Model Process for Removal of Caseins from Milk of    Transgenic Animals, BIOTECHNOL. PROG. 17:577-82 (2001).-   11. Prado S M, et al., (1999), Development and Validation Study for    the Chromatographic Purification Process for Tetanus Anatoxin on    Sephacryl S-200 High Resolution, BOLL CHIM FARM. 138(7):364-368.-   12. Porter, ed., HANDBOOK OF INDUSTRIAL MEMBRANE TECHNOLOGY, (Noyes    Publications, Park Ridge, N.J., (1998)) pp. 160-176.-   13. Ramachandra-Rao, H. G. et al., (2002) Mechanisms of Flux Decline    During Ultrafiltration of Dairy Products and Influence of pH on Flux    Rates of Whey and Buttermilk, DESALINATION, Vol. 144: 319-24.-   14. Reynolds, T. et al., (2003) Scale-Down of Continuous Filtration    for Rapid Bioprocess Design: Recovery and Dewatering of Protein    Precipitate Suspensions, BIOTECHNOL. BIOENG. August 20; 83(4):454-64-   15. Van Holten R. W. et al., (2003), Evaluation of Depth Filtration    to Remove Prion Challenge from an Immune Globulin Preparation, VOX    SANG. July; 85(1):20-4.-   16. Van Reis R., and Zydney A., Review, CURR OPIN    BIOTECHNOL., (2001) April; 12(2):208-11.-   17. Zeman, L. J. & Zydney, A. L. (1996), Microfiltration and    Ultrafiltration, in PRINCIPLES AND APPLICATIONS. (Marcel Dekker    ed.), New York.

UNITED STATES PATENTS AND INTERNATIONAL PATENTS INCORPORATED BYREFERENCE

-   1. Antonsen, K P et al., U.S. Pat. No. 6,194,553, PURIFICATION OF    ALPHA-1 PROTEINASE INHIBITOR.-   2. Jain M. et al., U.S. Pat. No. 4,351,710, FRACTIONATION OF PROTEIN    MIXTURES.-   3. Kothe et al., U.S. Pat. No. 4,644,056, METHOD OF PREPARING A    SOLUTION OF LACTIC OR COLOSTRIC IMMUNOGLOBULINS OR BOTH AND USE    THEREOF-   4. Sandblom R. M. et al., U.S. Pat. No. 4,105,547, FILTERING    PROCESS.-   5. Sherman, L. T. et al., U.S. Pat. No. 6,268,487, PURIFICATION OF    BIOLOGICALLY ACTIVE PEPTIDES FROM MILK.-   6. Udell, M. et al., European Patent No.: EP 1115745 (WO0017239),    PURIFICATION OF FIBRINOGEN FROM FLUIDS BY PRECIPITATION AND    HYDROPHOBIC CHROMATOGRAPHY.-   7. Van Reis R. M., et al., U.S. Pat. No. 6,555,006, TANGENTIAL-FLOW    FILTRATION SYSTEM.-   8. Van Reis R. M., et al., U.S. Pat. No. 6,221,249, TANGENTIAL-FLOW    FILTRATION SYSTEM.-   9. Van Reis R. M., et al., U.S. Pat. No. 6,054,051, TANGENTIAL-FLOW    FILTRATION SYSTEM.-   10. Van Reis R. M., et al., U.S. Pat. No. 5,490,937, TANGENTIAL FLOW    FILTRATION PROCESS AND APPARATUS.-   11. Van Reis R. M., et al., U.S. Pat. No. 5,256,294, TANGENTIAL FLOW    FILTRATION PROCESS AND APPARATUS.

1. A method for separating a protein of interest from a feedstream,comprising: (a) filtering said feedstream by a depth filtration processthat separates said protein of interest from said feedstream on thebasis of size; (b) performing a viral inactivation step that inactivatesat least 90% of viral particles or fragments; (c) performing an asepticfiltration step for the filtered feedstream; and, (d) clarifying theremaining material through additional downstream processing whereinmaterial that is retained by a filter during the depth filtrationprocess is trapped both upon a surface of the filter and within a matrixof the filter.
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. The methodof claim 1, wherein the feedstream containing the protein of interest iscomposed of the milk from a transgenic mammal.
 6. The method of claim 5,wherein casein proteins are disassociated from the rest of thefeedstream through the use of EDTA.
 7. The method of claim 6, furthercomprising wherein casein micelles are aggregated through the use of anammonium sulfate solution.
 8. (canceled)
 9. (canceled)
 10. (canceled)11. (canceled)
 12. The method of claim 1, wherein said protein ofinterest has a molecular weight of between 1 and 1000 kDa. 13.(canceled)
 14. (canceled)
 15. (canceled)
 16. (canceled)
 17. (canceled)18. (canceled)
 19. (canceled)
 20. (canceled)
 21. The method of claim 1,wherein said protein of interest is selected from the group consistingof proteins, immunoglobulins, polypeptides, antibodies, peptides andglycoproteins.
 22. (canceled)
 23. (canceled)
 24. (canceled) 25.(canceled)
 26. (canceled)
 27. (canceled)
 28. The method of claim 1,wherein said additional downstream processing comprises cationchromatography.
 29. The method of claim 1, wherein said additionaldownstream processing comprises size exclusion chromatography.
 30. Themethod of claim 1, wherein said additional downstream processingcomprises reverse phase chromatography.
 31. (canceled)
 32. (canceled)33. The method of claim 1, wherein the depth filtration process iscarried out at the protein of interest's isoelectric pH.
 34. The methodof claim 1, wherein the protein of interest is selected from the groupconsisting of alpha-1-antitrypsin, alkaline phosphatase, angiogenin,antithrombin, chitinase, extracellular superoxide dismutase, FactorVIII, Factor IX, Factor X, fibrinogen, glucocerebrosidase, glutamatedecarboxylase, human serum albumin, recombinant human albumin, insulin,myelin basic protein, lactoferrin, lactoglobulin, lysozyme, lactalbumin,proinsulin, soluble CD4, component and complex of soluble CD4, andtissue plasminogen activator.
 35. (canceled)
 36. (canceled) 37.(canceled)
 38. (canceled)
 39. A method for separating a protein ofinterest from a transgenic milk feedstream, comprising: (a) filteringsaid milk feedstream by a depth filtration process that separates saidprotein of interest from said feedstream on the basis of size; (b)performing a viral inactivation step that inactivates at least 90% ofviral particles or fragments; (c) performing an aseptic filtration stepfor the filtered feedstream; and, (d) clarifying the remaining materialthrough downstream processing; wherein material that is retained by afilter during the depth filtration process is trapped both upon asurface of the filter and within a matrix of the filter.
 40. (canceled)41. (canceled)
 42. (canceled)
 43. The method of claim 39, wherein saidmilk feedstream is diluted with an ammonium sulfate solution.
 44. Themethod of claim 39, wherein casein proteins are disassociated from therest of the feedstream through the use of EDTA.
 45. The method of claim44, further comprising wherein casein micelles are aggregated throughthe use of an ammonium sulfate solution.
 46. (canceled)
 47. (canceled)48. (canceled)
 49. (canceled)
 50. The method of claim 39, wherein saidprotein of interest has a molecular weight of between 1 and 1000kDa. 51.(canceled)
 52. (canceled)
 53. (canceled)
 54. (canceled)
 55. (canceled)56. (canceled)
 57. (canceled)
 58. (canceled)
 59. The method of claim 39,wherein said protein of interest is a selected from the group consistingof proteins, immunoglobulins, polypeptides, antibodies, peptides andglycoproteins.
 60. (canceled)
 61. (canceled)
 62. (canceled) 63.(canceled)
 64. (canceled)
 65. (canceled)
 66. (canceled)
 67. (canceled)67. (canceled)
 69. (canceled)
 70. (canceled)
 71. (canceled) 72.(canceled)
 73. (canceled)
 74. A method for isolating a protein ofinterest from a feedstream comprising: collecting a feedstream sample;diluting the feedstream sample with ammonium sulfate resulting in theprecipitation of contaminants, wherein the pH of the collectedfeedstream is above pH 5.0 subjecting the collected feedstream tomicrofiltration in a filtration unit with a mean pore size ranging from1.5 micron to 15 micron; subjecting the filtrate to tangential flowmicrofiltration with a limit of separation of 5,000 to 50,000 daltons.75. The method of claim 74, wherein the contaminants removed includemicelles and caseins.
 76. The method of claim 74, wherein the feedstreamis milk.
 77. (canceled)
 78. (canceled)
 79. (canceled)
 80. (canceled) 81.(canceled)
 82. (canceled)